Genetic Mechanisms Involved in the Generation of 
the Antibody Repertoire 
Frederick W. Alt, Ph.D. — Investigator 
Dr. Alt is also Professor of Biochemistry and Microbiology at Columbia University College of Physicians 
and Surgeons. He obtained his undergraduate degree in biology from Brandeis University and his Ph.D. 
degree in biological sciences from Stanford University, where he worked with Robert Schimke. He did 
postdoctoral work with David Baltimore at the Massachusetts Institute of Technology. His honors include 
the Irma T. Hirschl Career Scientist Award, the Searle Scholars Award, and the Mallinckrodt Scholar Award. 
WE are interested in the molecular mecha- 
nisms that underlie the generation of a spe- 
cific immune response. The mammalian immune 
system functions through complex interactions 
between various cells and their products. Cells 
that effect specific immunological responses fall 
into two general categories: B lymphocytes that 
mediate humoral immunity (i.e., production of 
antibodies against foreign antigens) and T lym- 
phocytes that mediate cellular immunity (e.g., 
foreign graft rejection) . B and T lymphocytes that 
actively fight infections and other diseases are 
generated in two general stages (primary and 
secondary) . 
During primary stages, stem cells proceed 
through a developmental program that ultimately 
leads to the generation of a multitude of individ- 
ual B or T lymphocyte clones (each clone is an 
essentially identical set of cells derived from a 
common parent). Each set of clonal cells ex- 
presses a novel receptor on its surface that will 
recognize a unique set of antigens. 
The secondary phase of lymphocyte differen- 
tiation results when a lymphocyte meets a foreign 
antigen (e.g., a bacterial cell surface component) 
that is recognized by its surface receptor; this 
stimulates the lymphocyte to divide and mature 
into an effector cell. For B lymphocytes, this mat- 
uration event involves secretion into the blood- 
stream of its specific receptor molecule; this se- 
creted product is the antibody. 
The ability of the immune system to respond 
specifically to a vast array of antigens results in 
substantial part from the unique organization of 
the genes that encode antigen receptor proteins. 
Unlike most genes, antigen receptor genes are not 
inherited intact from our parents. Instead, these 
genes are encoded in cassettes (gene segments) 
in the germline and are assembled into complete 
genes only during the somatic differentiation of 
lymphocytes. Because there are many individual 
cassettes that encode various portions of antibod- 
ies and because these can be put together in 
various combinations or in various ways, the 
body can randomly assemble a vast array of differ- 
ent antibody genes from a limited amount of ge- 
netic material. 
Much of our work is aimed at determining the 
genetic mechanisms by which antibody genes are 
assembled from their basic cassettes, the role of 
the gene assembly process in the generation of 
antibody diversity, and the mechanisms that regu- 
late this gene assembly process and ensure that it 
occurs only in appropriate cell types. We are also 
working on the elucidation of molecular signals 
that control the various steps of B lymphocyte 
differentiation. 
One experimental system employed in our 
studies involves the generation of cell lines that 
undergo the same types of antibody gene assem- 
bly events in culture dishes that normal develop- 
ing immature lymphocytes undergo in the ani- 
mal. In particular, we have devised and 
employed assays in which unrearranged antibody 
gene segments are isolated from normal mouse or 
human chromosomes and then propagated 
(cloned) in bacteria; we refer to these cloned 
molecules as "recombination constructs." Var- 
ious types of recombination substrates can be in- 
troduced into the permanent cell lines to ask 
mechanistic and regulatory questions about anti- 
body gene assembly. These studies have allowed 
us to clarify many mechanistic aspects of this 
gene assembly process and to devise schemes for 
how the assembly process is regulated. For exam- 
ple, we were able to show that both antibody 
genes and the analogous genes in T lymphocytes 
(encoding the T cell receptor) are assembled by 
the same general recombination machinery. 
A second general system that we employ to ana- 
lyze control of the recombination mechanism is 
the transgenic mouse. By injecting purified ge- 
netic material into fenilized mouse eggs and then 
implanting them into foster mothers, it is possi- 
ble to make mice that carry the foreign DNA in 
their germline. We have insened recombination 
substrates that contain experimentally manipu- 
lated antibody gene cassettes (or T cell receptor 
gene cassettes) into the mouse genome and asked 
questions about the genetic and cellular mecha- 
nisms that regulate assembly. These studies al- 
lowed us to extend our studies on clonal cell 
lines to identify more precisely several genetic 
